Project Abstract The long-term goal of this application is to understand the working mechanism of a supramolecualr machinery, termed divisome, in carrying out bacterial cell division. The divisome is composed of more than thirty proteins that are orchestrated to assemble and function at the correct time and space to ensure successful cytokinesis. Understanding key rate-limiting steps in this process and how different proteins coordinate with each other is important to provide potential new antimicrobial targets for treating bacterial infections. In the past a major focus of the field is on the role of the essential component of the divisome, a ring-like structure (Z-ring) formed by the tubulin homolog protein FtsZ, in generating a mechanic force to constrict the membrane actively. The roles of the other two components of the divisome, septal cell wall peptidoglycan (PG) synthesis machinery and chromosome segregation machinery, are thought to follow the active contraction of Z-ring. Recently accumulating evidence suggests that the Z-ring may not be the main force generator but a key regulator/mediator, and that cell wall synthesis and chromosome segregation machineries have larger roles in driving septum closure than Z-ring contraction. The project described here will examine this hypothesis using a combination of single-molecule imaging, genetic, biochemical and structural methods. The first Aim is to determine the role of the Z-ring in the spatiotemporal organization of septal PG proteins. The dynamics and organizations in wild-type FtsZ cells will then be compared to those in mutant cells harboring Z-rings with altered structures, dynamics, GTPase activity, and protein-proteins interactions. The second Aim is to determine the role of septal PG synthesis in driving septum closure. Septal PG synthesis will be systematically perturbed using targeted mutations and drug treatments, and the corresponding time-dependent septum closure rate, septal PG incorporation rate, septa morphology and composition will be measured using a variety of single-molecule imaging and biochemical methods. The third Aim is to determine the structural basis responsible for the coordination between chromosome segregation and septum closing. Molecular interfaces between a suit of proteins that interact with each other and anchor the Z-ring to the chromosome will be determined using genetic, biochemical, computational and structural methods. The expected outcomes of the project are: (1) a high-resolution structural and dynamic model of the full E. coli divisome, (2) a redefined roles and relative contributions of the three major divisome components in cytokinesis, (3) molecular insight into the working mechanism of the divisome as a whole, and (4) a set of innovative imaging- based tools and assays enabling bacterial cell biologists.